The overall objective of the proposed research is to investigate the biochemical properties, inborn errors and molecular genetics of three human heme biosynthetic enzymes, Delta-aminolevulinic acid dehydratase (ALA-D), porphobilinogen deaminase (PBG-D) and uroporphyrinogen III-cosynthase (URO III-CoS). Using methods developed in this laboratory, milligram quantities of human ALA-D, PBG-D and URO III-CoS will be purified to homogeneity for 1) characterization of their physicokinetic properties and reaction mechanisms, 2) production of polyclonal and monoclonal antibodies, and 3) determination of their N-terminal and peptide fragment amino acid sequences. Monoclonal antibodies will be used as affinity ligands for the rapid purification of each enzyme and to characterize the recently recognized human ALA-D polymorphic isozymes and the defective human enzymes in acute intermittent porphyria and congenital erythropoietic porphyria. The effect of lead and other metals on the physicokinetic properties of the ALA-D isozymes also will be determined. The clinical, pathologic and biochemical abnormalities in the newly discovered feline analogue of human acute intermittent porphyria will be characterized. In addition, this animal model will permit the unique opportunity to evaluate the pathogenesis of porphyric attacks, the porphyrinogenic potential of various drugs, as well as the biochemical effectiveness of therapeutic modalities (e.g., hematin, folate, glucose). Recombinant DNA techniques will be employed to isolate full length cDNA and genomic sequences encoding the three human heme biosynthetic enzymes and feline PBG-D for 1) characterization of their structure and organization, and 2) investigation of the molecular nature of the genetic defects in these porphyric disorders. Somatic cell and in situ hybridization techniques will be used to assign the structural genes for the human enzymes to specific sub-chromosomal regions. Using selected vectors, gene transfer experiments designed to correct the metabolic defect will be conducted in the feline porphyric model. These studies should provide increased understanding of the biochemical and molecular genetic defects and the basis for precise diagnosis and effective therapy of these human porphyrias.